2,8-Disubstituted-1,5-naphthyridines as Dual Inhibitors of Plasmodium falciparum Phosphatidylinositol-4-kinase and Hemozoin Formation with In Vivo Efficacy

Structure–activity relationship studies of 2,8-disubstituted-1,5-naphthyridines, previously reported as potent inhibitors of Plasmodium falciparum (Pf) phosphatidylinositol-4-kinase β (PI4K), identified 1,5-naphthyridines with basic groups at 8-position, which retained Plasmodium PI4K inhibitory activity but switched primary mode of action to the host hemoglobin degradation pathway through inhibition of hemozoin formation. These compounds showed minimal off-target inhibitory activity against the human phosphoinositide kinases and MINK1 and MAP4K kinases, which were associated with the teratogenicity and testicular toxicity observed in rats for the PfPI4K inhibitor clinical candidate MMV390048. A representative compound from the series retained activity against field isolates and lab-raised drug-resistant strains of Pf. It was efficacious in the humanized NSG mouse malaria infection model at a single oral dose of 32 mg/kg. This compound was nonteratogenic in the zebrafish embryo model of teratogenicity and has a low predicted human dose, indicating that this series has the potential to deliver a preclinical candidate for malaria.


■ INTRODUCTION
Malaria, a parasitic disease caused by the protozoan Plasmodium and transmitted to humans via the female Anopheles mosquito vector, remains a serious health problem, particularly in sub-Saharan Africa.Globally, there were an estimated 247 million malaria cases in 2021, with the African region accounting for 95% of cases. 1 The rollout of the RTS,S/ AS01 malaria vaccine in areas of moderate to high malaria transmission, along with several other initiatives to control and eradicate the disease, brings new hope for the eradication of malaria. 2 A new generation of antimalarial medicines is needed to combat the increasing drug resistance observed for artemisinin-based combination therapies, 3 which form the backbone of the treatment for life-threatening malaria caused by Plasmodium falciparum (Pf), the most problematic of the Plasmodium species.Ideally, new candidates would be affordable, well-tolerated, rapidly efficacious, have a low risk of resistance development, be long-acting, and remain at a sufficiently high concentration for long enough to completely clear parasites from patients with a single dose. 4reviously, we reported 2,8-disubstituted naphthyridines as potent inhibitors of Pf phosphatidylinositol-4-kinase β (PI4K), with the frontrunner compound 1 (Table 1) from the series showing 80% reduction in parasitemia at 4 × 50 mg•kg −1 in the humanized Pf NSG mouse malaria infection model. 5nadequate physicochemical properties, such as low aqueous solubility, high in vivo clearance, and poor selectivity against human phosphoinositide kinases required further optimization of the series to identify a preclinical candidate.During hit-to-Table 1. Exploration of Basic Amines at the Naphthyridine 8-Position a Inhibition of purified recombinant PvPI4K was determined in the presence of 10 μM ATP and ADP formation was quantified using the ADP-Glo Kinase assay kit.Mean IC 50 values ± standard deviation (SD) were calculated based on N ≥ 2 independent experiments, each with technical duplicates.ND: Not determined.b IC 50 values were determined using the parasite lactate dehydrogenase assay over 72 h as described, with means calculated from 2 independent experiments, each with 3 technical repeats.
the saturation or fraction sp 3 (Fsp 3 = number of sp 3 hybridized carbons/total carbon count) is known to improve solubility and reduce off-target promiscuity of molecules, reducing attrition during clinical development. 9,10Hence, to increase saturation in the molecules, several N-linked saturated heterocyclic rings were introduced at the C8-position with 3methylsulfonyl phenyl ring at the 2-position (Table 1).
The 3-methylsulfonyl substituent on the C2-phenyl ring retains potency, similar to the 3-sulfonamide substituent in 1 and is predicted to form strong hydrogen bonding (HB) interactions with the Ser1362 and Lys825 amino acid residues in the ribose pocket of the Plasmodium PI4K active site (refer to the section on kinase selectivity for more detailed binding interaction discussions). 6Compounds 8 and 9 with basic 3piperidine and 3-(R)-aminopyrrolidine rings, respectively, retained NF54 activity within 2-to 3-fold to that of 1 despite a substantial loss (21-to 28-fold) in PvPI4K enzyme inhibitory activity suggesting the contribution of additional MoAs driving the antiplasmodial activity of these two compounds.This result is consistent with the general observation that adding basic nitrogen enhances antimalarial activity for several unrelated scaffolds. 11The high solubility and potent antiplasmodial activity motivated further exploration of basic substituents at the naphthyridine 8-position.N-Acetylation or urea formation on the −NH 2 group of 9 (compounds 10 and 11, respectively) led to a 6-to 9-fold loss of NF54 activity, demonstrating the critical contribution of the basic N in maintaining higher antiplasmodial activity.Compounds 12 and 13 with the 4-CF 3 -3-pyridyl group at the C2-position of the naphthyridine ring showed ∼2-fold better antiplasmodial activity compared to the corresponding 3-methylsulfonyl matched pairs 8 and 9, respectively despite the comparable PvPI4K potency for 12 and a 6-fold loss in PvPI4K potency for 13.Compound 14, the S-3-aminopyrrolidine enantiomer of 13, maintained PvPI4K inhibition and NF54 activity similar to 13, suggesting the minimal influence of chirality on the antiplasmodial activity at this position.Similarly, enantiomer pair 15 and 16 with S-and R-3-aminopyrrolidine respectively, attached via the exocyclic NH at the C8-position of the naphthyridine ring showed comparable NF54 activity (IC 50 70 and 86 nM, respectively).Interestingly, the reversal of the attachment point to the naphthyridine core from the ring N of pyrrolidine as in 13 and 14 to the exocyclic NH improved PvPI4K potency by 7-to 18fold for both 15 and 16, although this did not lead to a corresponding improvement in antiplasmodial activity.
Compound 17, with a 4-aminopiperidine substituent at the 8-position attached via the exocyclic NH, showed potent NF54 activity (IC 50 = 40 nM) with a PvPI4K IC 50 = 53 nM and retained activity against K1 within 2-fold (IC 50 = 76 nM).Reversal of the attachment point to ring N of the piperidine ring led to 18, with a 10-fold loss in NF54 and K1 activity, whereas changing the "NH" linker to "O," as in 19, led to a 100-fold loss in NF54 activity.
SAR Exploration at the Naphthyridine 8-Position with Aminopiperidines.Since 17 showed favorable pharmacokinetic properties and kinase selectivity (discussed below) along with potent antiplasmodial activity, further SAR exploration was focused on subtle changes to the 4-piperidine substituent for optimization of potency and other properties.Compound 20, with a methylene unit inserted between the NH and piperidine ring, retained NF54 activity (IC 50 = 65 nM) but was 5-fold less active against the K1 strain (IC 50 = 350 nM) (Table 2).This compound also showed a substantial reduction in PvPI4K inhibitory activity (IC 50 = 1505 nM) compared to 17. N-methylation of the piperidine ring N (compound 21) retained NF54 activity (IC 50 = 52 nM) and moderate PvPI4K inhibition (IC 50 = 175 nM) similar to 17 with a K1 IC 50 (162 nM) within 3-fold of the NF54 IC 50 .
Larger hydrophilic or hydrophobic substituents on the piperidine ring N were not well tolerated, e.g., compound 22 with N-(2-hydroxyethyl)piperidine showed a 6-fold loss in potency against NF54 and PvPI4K whereas 23 with hydrophobic N-(2,2-difluoroethyl)piperidine showed >20-fold loss in NF54 activity even though it retained PvPI4K inhibitory activity similar to 17.The addition of a methyl substituent at the piperidine 3-position (compound 24) was well tolerated for NF54 activity and PvPI4K inhibition with good aqueous solubility.Compound 24 is a diastereomeric mixture and will require resolution to determine if there are significant differences in the potency and properties of individual diastereomers.Compounds 25 and 26 with dimethyl and cyclopropyl substituents at the 2-position of the piperidine ring, respectively, showed NF54 activity comparable to 17 but with a significant loss in PvPI4K potency.Compounds 27 and 28 with the −OH group at the C3-position of the piperidine, with a respective cis and trans geometric relationship of the −NH and −OH groups, retained NF54 activity within 3-to 4fold of 17 but showed a 7-and 18-fold lower PvPI4K potency relative to 17. Separation of enantiomers of both 27 and 28 would be required to determine if there are significant differences in the potency of the respective enantiomers.
SAR Exploration at the Naphthyridine 2-Position.The SAR scope with various 3-pyridyl substituents at the naphthyridine 2-position was investigated while keeping the N-methylaminopiperidine constant at the 8-position (Table 3).Removal of the CF 3 -group (compound 29) retained PvPI4K inhibitory activity but led to a 10-fold decrease in NF54 activity relative to the matched pair 21.The better antiplasmodial activity of 21 may be attributed to more potent inhibition of the other potential target compared to 29 or accumulation in high concentrations in the target-relevant compartment in the parasite due to better permeability Table 2. Exploration of NH-Linked Piperidine Derivatives at the Naphthyridine 8-Position a Inhibition of purified recombinant PvPI4K was determined in the presence of 10 μM ATP and ADP formation was quantified using the ADP-Glo Kinase assay kit.Mean IC 50 values ± standard deviation (SD) were calculated based on N ≥ 2 independent experiments, each with technical duplicates.b IC 50 values were determined using the parasite lactate dehydrogenase assay over 72 h as described, with means calculated from 2 independent experiments each with 3 technical repeats.
Table 3. SAR Exploration of Pyridyl Substituents at the Naphthyridine 2-Position a *3R-aminopyrrolidine at 8-position; Inhibition of purified recombinant PvPI4K was determined in the presence of 10 μM ATP and ADP formation was quantified using the ADP-Glo Kinase assay kit.Mean IC 50 values ± standard deviation (SD) were calculated based on N ≥ 2 independent experiments, each with technical duplicates.b IC 50 values were determined using the parasite lactate dehydrogenase assay over 72 h as described, with means calculated from 2 independent experiments each with 3 technical repeats.c SD not determined.Table 4. Naphthyridine Core Variations a Inhibition of purified recombinant PvPI4K was determined in the presence of 10 μM ATP and ADP formation was quantified using the ADP-Glo Kinase assay kit.Mean IC 50 values ± standard deviation (SD) were calculated based on N ≥ 2 independent experiments, each with technical duplicates.b IC 50 values were determined using the parasite lactate dehydrogenase assay over 72 h as described, with means calculated from 2 independent experiments, each with 3 technical repeats.
imparted by the lipophilic CF 3 group.Moving the CF 3 from the C4 to C2 position on the pyridine ring (compound 30) relative to 21 or adding a methyl group (2-methyl-6-CF 3 -3pyridine, compound 31) led to a 5-to 10-fold decrease in NF54 activity while retaining the PvPI4K inhibitory activity.A 3-and 8-fold loss in NF54 activity was observed for 32 and 33 when the hydrophobic CF 3 was replaced with more polar CN and CONHCH 3 substituents, respectively.Both compounds showed relatively higher K1 IC 50 values, particularly 33 (IC 50 > 6000 nM), suggesting susceptibility to the P. falciparum chloroquine resistance transporter (Pf CRT) that is thought to transport weak bases out of the digestive vacuole membrane. 12eplacing 4-CF 3 with 4-OCH 3 in 34 retained PvPI4K inhibition (IC 50 = 111 nM) but showed 2.5-fold lower NF54 activity (IC 50 = 225 nM) compared to matched-pair 16.Further extension of 4-OCH 3 as a 3-hydroxylpropyloxy substituent in 35 improved PvPI4K inhibition by 6-fold (IC 50 = 19 nM) compared to 34, likely by gaining solvent interactions with the polar −OH group in the ribose pocket of the Pf PI4K active site, but without any improvement in NF54 activity.All these observations indicated limited structural scope at the C2 position of the naphthyridine core for improving antiplasmodial activity, even though PI4K inhibition could be optimized by adding polar groups.
The overall SAR study, revealing a poor correlation between in vitro PvPI4K inhibition and whole-cell activity, suggested an additional MoA for the naphthyridines with the C8-position basic substituents.Hence, representative compounds from the series with similar NF54 activity but a distinct difference in PI4K potency were selected along with 1 for more detailed MoA studies.
Screening against P. falciparum PI4K Mutants.Compounds from the series were tested against PI4K inhibitor-resistant Dd2-derived lab mutants generated in response to the PI4K inhibitor KAI407 and a mutant with a second copy of PI4K (Dd2_PI4K_CNV) to evaluate whether PI4K remains the primary MoA. 7Known PI4K inhibitor KDU691 was used as a reference compound in the experiment (Table 5). 7It can be seen that the reference compound shows less activity against the mutants than the wild-type strain, as would be expected for a PI4K inhibitor.The parent compound 1 shows significantly higher IC 50 values in two of three mutants including the mutant with the additional copy of PI4K (Dd2_PI4K_CNV), again consistent with PI4K being the primary MoA.The observation that 1 is equipotent in the reference strain and one of the PI4K double mutants shows that the mutations generated by the reference compound might not affect the binding of other PI4K inhibitors from different chemical classes.This can also be seen in Table 6, where 1 shows some cross-resistance to the PI4K mutant DD2_048, 13 which was isolated after drug pressure with MMV390048, a PI4K inhibitor from another chemical class.Conversely, 14, 17, 27, and 28 showed no substantial increase in IC 50 against any of these mutants relative to their IC 50 in the  wild-type Dd2, providing supporting evidence for a change in the MoA of compounds with basic substituents.Cross-Resistance Profiling against Lab-Raised Resistant Lines and Field Isolates.Compounds 1 and 17 were also tested against several field isolates as well as lab strains derived by raising resistance to various in-development antimalarial drug candidates (Table 6).Compound 1 showed a marked 2.4-fold difference in IC 50 against the PI4K mutant (Dd2_048).On the other hand, 17 was equipotent to the Dd2 lab-raised mutants tested in these panels, including the PI4K mutant.Hence, the data supports a primary PI4K MoA for 1 but not for 17, and there is no evidence for a DHODH, Pf CARL, Cytochrome b, or translation elongation factor 2 MoA associated with the other drug candidates.Compounds 1 and 17 showed 0.7−1.0-foldand 1.3−2.5-folddifferences in IC 50 against field isolates compared to the NF54 wildtype parasites, respectively.Attempts to raise mutants to 14 following sustained drug pressure at 3 × IC 90 for 60 days in vitro were not successful (data not shown).Conversely, a high propensity for resistance has been seen after applying sustained drug pressure with previous PI4K inhibitors, such as MMV390048. 3These data provide further evidence that this series has moved away from PI4K inhibition as the primary mode of action, and it is thought that perhaps targeting an additional secondary process alongside PI4K inhibition has resulted in the lowered risk of resistance with 14.
Effect of PI4K Knockdown on Parasite Susceptibility to Inhibitors.To test the role of Plasmodium PI4K inhibition in the MoA of this series, representative compounds 1, 14, 16, and 17 were profiled against a Plasmodium PI4K conditional knockdown (cKD) line 18 in an asexual blood stage assay (Figure 1 and Table S5).In this engineered parasite line, translation of PI4K is controlled by anhydrotetracycline (aTc)  S4).PI4K inhibitor MMV390048 was included as a positive control.using the TetR (Tet repressor protein)/DOZI (development of zygote inhibited)-RNA aptamer module. 19,20In the presence of high aTc concentration PI4K translation occurs, whereas low aTc concentration results in reduced PI4K expression (knockdown of PI4K).Plasmodium PI4K inhibitor MMV390048 was included as a positive control.As expected, knockdown of PI4K led to increased parasite sensitivity to MMV390048, as exhibited by a leftward shift in the dose− response curve (Figure 1a) corresponding to a 6.9-fold decrease in IC 50 relative to control conditions.Similarly, knockdown of PI4K also resulted in increased sensitivity to Compound 1 (7.6 fold decrease in IC 50 , Figure 1b), confirming that PI4K is the primary target for this compound.In contrast, the knockdown of PI4K did not result in significant differential parasite susceptibility to compounds 14, 16, or 17 (Figure 1c− e), further supporting the hypothesis that PI4K inhibition is not the primary (or only) MoA for these 8-position basic derivatives.
Inhibition of Hemozoin Formation.During the asexual blood stage propagation, the Plasmodium parasite develops from merozoite through ring and trophozoite to a mature schizont before erupting from the RBC and invading new RBCs, causing fever and chills every 2−4 days.The metabolically active trophozoite digests large quantities of RBC hemoglobin (Hb), utilizing the globin part for its nutritional requirements.Heme released as a byproduct of Hb degradation is detoxified by crystallization into an inert, insoluble pigment known as hemozoin. 21Chloroquine and related quinoline-containing drugs inhibit the formation of hemozoin, resulting in rapid plasmodial death from the accumulation of toxic heme. 22On the other hand, disruption and reduction of hemoglobin-derived peptides is the typical signature observed for Pf PI4K inhibitors. 23Coupled with structural similarities between some kinase and hemozoin formation inhibitors vis-a-vis the presence of multiple heteroaromatic rings, planar structures, and basic nitrogens, this prompted us to investigate inhibition of hemozoin formation as a potential contributing MoA for naphthyridine derivatives with basic C8 substituents.This potential was assessed initially by measuring the ability of the compounds to interfere with the formation of synthetic hemozoin, β-hematin (βH), in vitro in a cell-free detergent-mediated Nonidet P-40 (NP-40) assay (Table S1). 24Most of the 8-position basic analogs, e.g., compounds 14, 16, 17, 20, 21, 23, and 36 were potent inhibitors of βH in the NP-40 assay (βH IC 50 < 60 μM) whereas compound 1 showed no inhibition at the limit of assay detection (βH IC 50 > 500 μM).To confirm that the βH-active compounds are bona fide inhibitors of hemozoin formation in whole-cell parasites, 17 was tested in a cellular heme fractionation assay to measure free Hb, heme and hemozoin profiles when synchronized ring stage parasites were treated with increasing concentrations of the compound. 25In these experiments, 17 showed a significant dose-dependent increase in the levels of heme and hemozoin relative to the untreated control, confirming hemozoin inhibition in the parasite cells by the compound (Figure 2).The data indicates that inhibition of hemozoin formation is likely the primary MoA contributing to the antiplasmodial activity of 17.
Parasite Reduction Ratio (PRR).The relief of clinical symptoms of malaria is linked to rapid clearance of parasite load in patients during treatment.This property is desirable for any antimalarial treatment, and thus the killing kinetics of 1 and 17 were determined using the PRR assay as described. 26ompound 1 showed a log PRR value of 1.7 (Figure S1), which is classed as fairly slow-acting.In addition, there was a lag phase of 24−48 h before the onset of action, similar to the existing antimalarial compound pyrimethamine and consistent with the trend seen with compounds with known PI4K inhibition properties, such as MMV390048 and UCT943. 27ompound 1 shows a parasite clearance time of 69.8 h.Conversely, 17 had log PRR values of 3.9 (Figure S1), classed as fast-acting, similar to chloroquine (log PRR = 4.1).These data correlated with the extrapolated parasite clearance times, which are around 40 h for 17, compared to 36.8 h with CQ.Additionally, 17 did not show any delay before the onset of action.These data support there being different MoAs for the C8-position basic analogs relative to 1.
Gametocytocidal Activity.Killing of gametocytes circulating in the bloodstream of patients can limit transmission of malaria, because gametocytes need to move into a mosquito host during a blood meal to continue the parasite life cycle.Previously described PI4K inhibitors such as KDU691 and MMV390048 have shown activity against the sexual blood stages of the parasite, with low nanomolar potency against gametocytes, similar to the potency observed against asexual blood stage parasites (∼5-fold difference in activity)., 727 Compounds 1 and 17 showed limited activity against immature and late-stage gametocytes, 13 with 17 showing improved activity over 1 against early stage gametocytes.Compound 1 gave an IC 50 of 2.22 and 2.24 μM against earlyand late-stages, respectively, and 17 gave values of 0.59 and 2.20 μM, respectively.The loss in activity of 17 against mature, but not immature gametocytes, supports the additional mode of action of 17 inhibiting hemozoin formation aside of PI4K inhibition, with immature gametocytes still sustaining hemozoin formation, a process that is lost in mature, stage V gametocytes. 28This data is consistent with the loss of activity of hemozoin inhibitors like chloroquine against mature gametocytes. 29 Gamete Exflagellation Inhibition.Gamete formation is an essential part of the sexual stages of the parasite life cycle and occurs in the mosquito midgut after ingestion of male and female gametocytes by the mosquito during feeding.Blocking this process prevents the life cycle from continuing and is thus a potential mechanism of limiting disease transmission.Previously described PI4K inhibitors such as MMV390048 are known to inhibit gamete formation, as shown using the dual-gamete formation assay. 27Compounds 1 and 17 were tested at a concentration of 2 μM in the exflagellation inhibition assay to determine if compounds interfered with the formation of P. falciparum male gametes. 30Compound 1 showed 60% inhibition of exflagellation at a concentration of 2 μM, similar to MMV390048. 27Conversely, 17 showed only 11% inhibition at 2 μM in line with its weaker PvPI4K inhibitory activity compared to 1.This again provides additional support to Hz inhibition as a main contributor to the MoA of 17, as inhibitors of Hz formation are not active against male gametes.

Plasmodium falciparum Liver Schizont Inhibition
Assay.Compounds 1 and 17 were tested for their ability to inhibit schizont formation from sporozoite infection in cultured primary human hepatocytes as described, 31 which shows the potential of the compound for prophylactic use by preventing the establishment of the asexual blood stage after infection via a mosquito bite.Numerous PI4K inhibitors described previously have shown potent activity in this assay. 32lthough 1 showed substantial inhibition with an IC 50 of 93 nM, the basic derivative 17 showed no activity at 1 μM and only 59 ± 15% inhibition at 10 μM (n = 2), consistent with a change in MoA.
Selectivity against Human Kinases.A few representative naphthyridines, 1, 14, 16, and 17 were screened for off-target activity against the human orthologue HuPI4Kβ, the related phosphoinositide kinase HuPI3Kα and protein kinases HuMAP4K4 and HuMINK1 (Table 7).These human kinases were identified as key off-targets potentially linked to the embryo-fetal toxicity (teratogenicity) observed in rats for the Plasmodium PI4K inhibitor MMV390048 that was evaluated in clinical studies. 33Compound 1 showed potent inhibition of HuPI3Kα and HuPI4Kβ enzymatic activity at 1 μM whereas the compounds 14, 16, and 17 showed very weak or no inhibition of HuPI4Kβ, HuMAP4K4, and HuMINK1 (IC 50 > 10 μM) and weak inhibition of HuPI3Kα in the micromolar range.Overall, introducing the basic substituents at the C8position reduced the kinase inhibition by the compounds in  the series substantially while retaining the potent NF54 activity attributed to a MoA other than (or in addition to) PI4K inhibition.This improved off-target activity profile could potentially avert the risk of embryofetal toxicity for these compounds, although the compounds need to be screened against a larger panel of human kinases to identify any other selectivity issues.
Structure-Based Rationale for the Selectivity against Human Lipid Kinases.This series of 2,8-disubstituted 1,5naphthyridines was docked into a previously reported Pf PI4K homology model using GLIDE. 6The predicted binding mode of a 2,8-naphthyridine series within the ATP-binding site of the Pf PI4K homology model has been described previously. 5In this model, the naphthyridine N5 is predicted to interact with the kinase hinge, accepting a H-bond from the backbone amide of V1357.The substituents at the naphthyridine 8-position extend toward the catalytic pair K1308 and D1430, forming polar interactions with either of these two residues.The 2position substituent extends into the ribose pocket, with the possibility of forming hydrogen bonds with the S1362 and S1365 residues.Docking observations were compared with the in vitro PvPI4K IC 50 values to rationalize changes in Plasmodium PI4K potency.The molecular basis for off-target HuPI4Kβ inhibition for this series was rationalized by docking compounds into the HuPI4Kβ crystal structure (PDB ID: 4D0L).
Compound 17 is predicted to interact with the Pf PI4K ATP site in accordance with the previously described binding mode for this series (Figure 3A).With the prerequisite H-bond interaction between the hinge and the heteroaromatic naphthyridine N5, the 8-position piperidine occupies a smaller space in the catalytic region than other compounds in the series with larger substituents.It is therefore able to form a saltbridge interaction with the catalytic D1430 without extending too deep into the catalytic region, avoiding a clash with the positively charged K1308.The 2-position 4-CF 3 -3-pyridyl substituent extends into the ribose pocket without any clashes with the two serine residues in this subsite, unlike related phosphoinositide kinases, which often feature larger residues at these positions.
When docked into the ATP-binding site of the HuPI4Kβ crystal structure, 17 displayed a binding pose analogous to the one observed in Pf PI4K with the same hinge, catalytic site and ribose pocket interactions (Figure 3B).The key difference between the two binding sites is that the ribose pocket of HuPI4Kβ contains residue Q606 in a locus analogous to S1362 of Pf PI4K.The larger Q606 is predicted to clash with the 4-CF 3 -3-pyridyl of 17 and disrupt the hinge binding H-bond with the 5-position naphthyridine N.This brings the basic pyrrolidine closer to the catalytic K549, creating an electronic clash between two positively charged groups.This clash may account for no inhibition observed below 10 μM against HuPI4Kβ.Conversely, Pf PI4K with a smaller and less bulky S1362 residue in this ribose pocket position is not predicted to clash with the CF 3 -pyridyl group of 17, allowing for an optimal hinge binding H-bond with the naphthyridine N, giving it enough distance from the catalytic K1308 to avoid an electronic clash.This provides a potential explanation for how 17 and related analogs in the series retain PvPI4K inhibition while reducing activity against HuPI4Kβ.A similar interaction of the 4-CF 3 -3-pyridyl group of MMV390048 with the ribose pocket has been hypothesized to contribute to the impressive 1000-fold selectivity toward Pf PI4K over HuPI4Kβ. 6n Vitro Teratogenicity Assessment by Zebrafish Embryo Assay.Acute toxicity and teratogenicity assays in zebrafish embryos have become valuable in vitro tools for assessing safety of new compounds in the early preclinical stages and may be used for filtering out compounds for progression to the resource-intensive preclinical animal safety models. 34Compound 17 was tested in a zebrafish embryo assay for potential teratogenic effects.Briefly, in this assay, the fertilized embryos of zebrafish are treated with serial dilutions of a compound to determine the lethal concentration (LC 50 ), a concentration at which 50% of larvae die at 96 h post fertilization and active concentration (AC 50 ) at which 50% larvae population show a teratogenic phenotype for a compound.A compound is categorized as a teratogen when the teratogenic effects are observed much below the lethal concentration.The teratogenic index (TI), defined as a ratio between LC 50 and AC 50 , of >3 for the most sensitive teratogenic phenotype would classify compound as a teratogen.Compound 17 with TI = 1.1 was categorized as a nonteratogen in this assay (Table 8).The data indicates that the series could deliver compounds with low risk of teratogenicity for further progression into the preclinical studies with the appropriate C8-position substituent.
hERG Inhibition.A large number of basic compounds are known to inhibit cardiac hERG channels, leading to QT prolongation with the potential to cause life-threatening cardiac arrhythmia. 35A few analogs from the basic naphthyridine series were screened for hERG inhibition in the QPatch automated patch clamp assay that employs CHO cells stably expressing the hERG channels (Table 9). 36mpound 17 showed a hERG IC 50 of 3 μM, which is aligned with the basic lipophilic nature of the compound (pK a 9.42, log D 0.81).Compounds 25 and 26 with dimethylpiperidine and cyclopropyl piperidine with reduced basicity (pK a 8.98 and 7.99, respectively) retained similar hERG IC 50 's, likely due to the higher lipophilicities of these compounds.However, hydroxypiperidine 27, having lower log D and pK a (pK a 8.99, log D = 1.02,) showed a significant reduction in hERG inhibition (hERG IC 50 24.5 μM) while retaining antiplasmodial activity (NF54 IC 50 = 140 nM) within 3-fold of 17.The data suggests that hERG inhibition by this class of compounds can be reduced by the introduction of polar substituents at  appropriate positions without significant loss of antiplasmodial activity.This observation will be useful in further optimization of the compounds while minimizing the hERG inhibition liability.Pharmacokinetic Studies.The in vitro metabolic stability of potent compounds was tested in mouse, rat, and human liver microsomes, where high metabolic stability was seen (Table 10).The compounds did not show significant inhibition of cytochrome P450 enzymes.Compounds 14, 16, 17, and 21 were tested for in vivo pharmacokinetic properties in mouse and showed significantly lower unbound clearance compared to 1, as well as moderate to high oral bioavailability.The low unbound clearance of 17 resulted in higher free drug concentration relative to 1 (freeAUC = 562.8and 13.6 min•μmol/L for 17 and 1, respectively, in mouse at a 10 mg/kg oral dose).Relating these values to the in vitro NF54 IC 50 's predicting a lower efficacious dose for 17 relative to 1. Compound 21, the N-methylpiperidine analog of 17, showed significantly improved oral bioavailability (F = 99%) compared to 17 (F = 40%), most likely due to its better intestinal permeability in line with its higher lipophilicity.Overall, 17 and 21 showed similar freeAUC's (oral dosing) and activities against Pf NF54.The former was chosen for efficacy studies due to its higher solubility and better activity against the K1 multidrug resistant P. falciparum strain.
In Vivo Efficacy.Compound 17 was tested in a humanized NSG mouse model of malaria by administering single ascending oral doses of 3, 10, 30, 50, and 100 mg/kg 3 days post infection. 37The blood samples from treated mice were screened for drug concentration and parasitemia every 24 h from day 3 until day 7 post infection to generate the pharmacokinetics and pharmacodynamic parameters, respectively (Figure 4).The compound showed dose and timedependent reduction in parasitemia with ED 90 , the effective dose in mg/kg that reduces parasitemia by 90% on day 7 following infection compared to the untreated control group, of 32 mg/kg with the corresponding oral exposure levels (AUC ED90 ) of 16.3 μM•h (Figure S2).The pharmacokinetics analysis (Figure 4B, Table S2) showed a dose proportional increase in oral exposure with doses of 30 mg/kg and above achieving significant free drug exposure above NF54 IC 50 .The data demonstrated much improved efficacy for 17 in accordance with improved pharmacokinetic properties compared to 1 that showed only 80% reduction in parasitemia at 4 × 50 mg/kg QD doses.ND >20, >20, 18, >20 >20, >20, >20, ND >20, >20, >20, ND ND Caco-2 P app A > B (10  Early Human Dose Predictions.Human dose prediction is increasingly recognized as an essential parameter in drug discovery to save critical time and resources.MMVSola is a tool that predicts human pharmacokinetics and dose required to clear malaria parasites from a patient. 38It does this by using the physicochemical and biological properties of a compound, together with the in vivo DMPK data and NSG EC 50 determined during the PK-PD modeling (Table S3).The tool predicts the human volume of distribution as the geometric mean of the unbound volume of distribution between preclinical species. 39The human clearance is calculated through extrapolation of human in vitro data using coefficients derived from in vitro and in vivo preclinical species data. 40The human dose is then calculated as the dose required to clear 12-log units of parasitemia and to maintain concentrations above the minimum parasiticidal concentration (MPC) for 7 days.The MPC is therefore defined as the lowest blood concentration resulting in the maximum parasiticidal effect.Compound 17 showed a low predicted human dose of 51 and 69 mg for a 9 and 12 log reduction in parasitemia, respectively (Table S4).In addition, 17 also has a predicted half-life in humans of greater than 120 h, indicative of the potential of the series to meet the long high-life criteria of a preclinical candidate.It is important to note the early human dose prediction was based on rodent PK only.An accurate human dose prediction would require PK in an additional higher species.

■ CONCLUSIONS
The described study identified a class of 2,8-disubstituted-1,5napthyridines with basic substituents at the 8-position of the naphthyridine ring and potent antiplasmodial activity that has emerged following previous SAR studies in the series.A subset of these compounds retained nanomolar Plasmodium PI4K inhibition but showed inhibition of hemozoin formation as the likely primary MoA contributing to their antiplasmodial activity.The compounds retained activity against clinical isolates, including drug-resistant strains.Basic substituents on the naphthyridine 8-position significantly improved aqueous solubility compared to previously reported analogs with neutral substituents.The basic naphthyridine substituents improved in vivo pharmacokinetic properties, resulting in much improved efficacy in the NSG mouse model of malaria with a single dose ED 90 of 32 mg/kg for a representative compound 17.These compounds showed minimal off-target inhibitory activity against the human phosphoinositide kinases and MINK1 and MAP4K kinases, which are potentially associated with the teratogenicity and testicular toxicity observed in rats for the Pf PI4K inhibitor clinical candidate MMV390048.Compound 17 was nonteratogenic in a zebrafish embryo assay and has a low predicted human dose based on the MMVSola predictions, indicating the potential of the series to deliver a late lead candidate.Additionally, the predicted long half-life of compound 17 and the apparently low risk of resistance seen with compound 14 make the series an attractive, with both these criteria being earmarked as essential for future drug candidates by the malaria research community.The SAR studies demonstrated in the manuscript would be helpful for further optimization of hERG inhibition of the compounds to identify a preclinical candidate from the series.
■ EXPERIMENTAL SECTION DMPK.All protocols for in vitro DMPK studies and mouse PK studies are available in the Supporting Information.Animal studies were conducted following guidelines and policies as stipulated in the UCT Research Ethics Code for Use of Animals in Research and Teaching, after review and approval of the experimental protocol by the UCT Senate Animal Ethics Committee (protocol FHS-AEC 013/ 032).
Modeling.All docking simulations were run on the Pf PI4K homology model published by Fienberg et al. or structures downloaded from the PDB.Protein structures were all processed using the Maestro GUI of the Schrodinger 2023−4 (Schrodinger Release 2023−4: Schrodinger, LLC, New York, NY, 2023.)software suite.Protein structures were prepared using the Schrodinger Protein Preparation wizard using default preprocessing, H-bond optimization and minimization settings.Crystallization artifacts were manually removed, and the residues with alternate positions were manually assigned.The prepared ligand binding site was then visually inspected for correct tautomer and H-bond assignment.
Docking grids were then prepared using the GLIDE Receptor Grid Generation tool with a grid centered upon previously docked ligands in the active site or the crystallized ligand from HuPI4Kβ. 41A hydrogen bonding constraint on the hinge H-bond donating amide (Pf PI4K−V1357, HuPI4Kβ−V598) was also set.
All ligands were prepared from SMILES using LigPrep with ionization states determined to a pH of 7 ± 1.0, calculated using the Epik and the OPLS4 atomic forcefield. 42The prepared ligands were then docked into the prepared docking grids using SP docking.Ligands that failed to dock with a plausible pose were redocked with higher precision with the top 10 poses retained.Every ligand for which a plausible docking pose was determined then had its binding energy scored using Prime MM-GBSA with the minimization radius set to full residues within 5 Å of the ligands using the VSGB implicit solvation model and the OPLS4 forcefield with the minimize sampling method.
All docking images were generated using open source PyMOL 2.5.0 (Schrodinger, LLC.2010.The PyMOL Molecular Graphics System, Version 2.5.0).
Chemistry.All commercially available reagents were purchased from Sigma-Aldrich or Combi-blocks.Unless otherwise stated, all solvents used were either anhydrous or analytical grade.Where stated, microwave synthesis was conducted using a Discover/Explorer12 microwave reactor from CEM Corporation.Column chromatography was performed using a Teledyne ISCO combi flash system in either normal (on prepacked Silicycle silica gel cartridges) or reverse phase (on prepacked Silicycle C18 cartridges) modes, while HPLC was performed on a Teledyne ISCO ACCQPrep HP150 system eluting a reverse phase (C18) solvent gradient with an appropriate solvent gradient (water and acetonitrile or methanol, with or without 0.1% formic acid). 1 H NMR spectra were recorded on a Bruker Spectrometer at 300 MHz. 13 C NMR spectra were recorded on a Bruker 400 or 600 MHz spectrometer.Chemical shifts are reported in parts per million (ppm) downfield from TMS as the internal standard.Coupling constants, J, are reported in Hertz (Hz).Standard acronyms representing multiplicity are used: br s = broad singlet, s = singlet, d = doublet, t = triplet, m = multiplet.Purity was determined using an Agilent 1260 Infinity binary pump, Agilent 1260 Infinity diode array detector (DAD), Agilent 1290 Infinity column compartment, Agilent 1260 Infinity standard autosampler, and Agilent 6120 quadrupole (single) mass spectrometer, equipped with ESI ionization source.All compounds tested for biological activity were confirmed to have ≥95% purity.LC purity analyses were performed using one of the following methods: Method 1: Using a Kinetex 1.7 μM C-18 column, (Mobile phase A: 10 mM buffer (Ammonium acetate/acetic acid) in H 2 O and Mobile phase B: 10 mM buffer (Ammonium acetate/acetic acid) in Methanol).All synthesized intermediates were characterized by liquid chromatography−mass spectrometry (LCMS), while final compounds were confirmed by LCMS and, at least, a 1 H NMR data.
General Synthesis Methods.Method A: To the intermediate 7 (1 equiv) in 1,4-dioxane (25 mg/mL) was added the appropriate amine (2 equiv.)and sodium tert-butoxide (1.5 equiv).The reaction mixture was degassed by bubbling nitrogen (N 2 ) through it.Tris(dibenzylideneacetone)dipalladium(0) (0.1 equiv) and tritertbutylphosphine (1 equiv) were added.The mixture was further degassed and heated at 110 °C for 18 h.The reaction mixture was filtered through a Celite pad.The crude filtrate was adsorbed onto silica gel and purified by normal phase column chromatography using a prepacked silica gel column on Combiflash, eluting in a gradient of ethyl acetate in petroleum ether, unless otherwise stated.

Figure 1 .
Figure 1.Effect of conditional knockdown (cKD) of PI4K on parasite sensitivity to (a) control compound MMV390048, (b) compound 1, (c) compound 14, (d) compound 16, and (e) compound 17, relative to control conditions in the presence of high aTc.Representative dose−response curves are shown where error bars represent ± SD for technical duplicates.Results were confirmed in N = 3 independent experiments (TableS4).PI4K inhibitor MMV390048 was included as a positive control.

Figure 3 .
Figure 3. Predicted binding mode of basic naphthyridines in Pf PI4K and HuPI4Kβ (A) Compound 17 docked into the ATP binding site of a Pf PI4K homology model.6The 5-position naphthyridine N accepts a hinge H-bond from the backbone amide of V1357.Potential π-stacking interactions are observed between the naphthyridine core and both Y1356 of the hinge and F827 of the P-loop region.The basic piperidine at 8position is positively charged and forms a salt bridge with the catalytic D1430; (B) Compound 17 docked into the crystal structure HuPI4Kβ (PDB ID: 4D0L).This pose focuses on the clash predicted between Q606 of the ribose pocket and the 4-CF 3 -3-pyridyl substituent on the naphthyridine 2-position.This clash creates a suboptimal geometry for the hinge binding H-bond with the backbone NH of V598 while bringing the basic piperidine closer to K549 where it experiences an electronic clash despite potential polar interactions with two proximal acids, explaining the loss of activity.

Figure 4 .
Figure 4. (A) In vivo therapeutic efficacy of 17 in the humanized NSG mice infected with P. falciparum Pf 3D7 0087/N9 cells and (B) the free plasma concentrations vs time in NSG mice following a single oral administration.(The red dashed line represents the in vitro NF54 IC 50 of 17.).

Table 6 .
Activity against Field Isolates and Dd2-Derived Lab Mutants Resistant to Known Antiplasmodials a

Table 7 .
Inhibition of Human Lipid Kinases a a <5% enzymatic activity remaining at 1 μM of the compound; IC 50 was not determined.ND: Not determined.